Consistent with the multiple objectives of PA-08-208, Pilot Studies in Pancreatic Cancer, we are proposing to develop a novel experimental model of human pancreatic cancer that will facilitate the identification of novel genetic and epigenetic aberrations underlying PanIN formation. The central hypothesis guiding this work is that by studying molecular and cellular changes at the very earliest time points following oncogenic Kras activation, even prior to the onset of morphologic PanIN formation, we will gain unique insight into the mechanisms underlying the true initiation of pancreatic cancer, in a manner that will allow effective chemoprevention and/or pharmacologic termination of PanIN progression. In pursuit of this goal, we are proposing to study the initiation and progression of pancreatic cancer with unprecedented temporal and spatial resolution. We are currently generating a new mouse model in which a lox-stop-lox-GFP::KrasG12D cassette encoding a GFP-KrasG12D fusion is knocked into the endogenous Kras locus. Unlike prior models, this approach will allow for the direct visualization and FACS-based isolation of specific cell populations in which oncogenic Kras has been activated, even prior to the onset of morphologic change. This will not only allow genetic, epigenetic and functional analyses to be performed with unprecedented temporal resolution, it will also allow for these analyses to be carried out on the level of individual cells, allowing an entirely novel view of cellular heterogeneity within both forming PanINs and invasive later lesions. To test the above hypothesis, we will pursue the following Specific Aims: First, we will visualize the earliest cellular responses to LSL-GFP::KrasG12D activation in the acinar and ductal compartments using a novel in vitro culture system, and will determine the signaling pathways mediating these responses; second, we will generate and compare high resolution temporal mapping of the pre-PanIN and PanIN transcriptomes following cell type-specific activation of LSL-GFP::KrasG12D in acinar and ductal lineages; and third, we will generate and compare high resolution temporal mapping of surface marker expression in individual cells following activation of LSL-GFP::KrasG12D in the acinar and ductal lineages. Together, these Aims will provide highly innovative and important insights into the earliest events in PanIN formation, as well as an initial glimpse at the onset of cellular heterogeneity during Kras-mediated neoplasia. PUBLIC HEALTH RELEVANCE: This application seeks funding to generate a novel murine model of human pancreatic cancer, in a manner that will allow the direct visualization, isolation and characterization of tumor cells with unprecedented spatial and temporal resolution.